The p53 tumor suppressor counters a wide range of stresses, including chemical mutagens, ionizing radiation, hypoxia, and nutrient deprivation, by activating growth arrest or apoptosis programs. All cancers must inactivate the p53 network at one or more of its nodes to progress. Most often, p53 is mutated such that it is inactivated in tumors. In other cases, regulators of p53 are altered instead of p53 itself. Frequently, the regulators that are altered in cancer cells participate in the normal destruction of p53 protein by the ubiquitin/proteasome system. The pathway of p53 destruction by the ubiquitin/proteasome system is complex, involving monoubiquitination, polyubiquitination, recognition by polyubiquitin-binding proteins, and recognition and degradation by the proteasome. p53 is monoubiquitinated by the E3 ligase MDM2, and work from the prior grant has shown that the p300/CBP coactivators are E4 enzymes catalyzing further polyubiquitination of p53 in the cytoplasm. Polyubiquitinated p53 can be recognized by the hHR23 family of proteasome adaptor proteins, which appear to shield p53 from proteasome destruction after cell stress such as DNA damage, but may also normally deliver p53 to the proteasome. hHR23 proteins, in turn, are recognized by the S5a subunit of the proteasome, which may be the gateway to the proteasome for p53, leading to its destruction. The Aims of this renewal application are to: 1. Characterize the structure and function for p53 polyubiquitination of a p300/CBP E4 activity 2. Investigate the role of p300/CBP E4 and Ub binding activities in p53 regulation 3. Investigate the role of hHR23 and S5a in p53 regulation A firm and complete understanding of the pathway of p53 destruction by the ubiquitin/proteasome system will increase our knowledge of how cancers form through disruption of this pathway, and also lead to the development of novel cancer therapeutics targeting this pathway. Such therapeutics hold the hope of reactivating p53 in those tumors harboring wild type p53, leading to the arrest or apoptosis of malignant cells within.